Molecular cloning of kuruma shrimp Marsupenaeus japonicus phosphopyruvate hydratase and its role in infection by white spot syndrome virus and Vibrio alginolyticus

Abstract

Phosphopyruvate hydratase (enolase) is a housekeeping gene for energy production in many eukaryotes. The 1914-base pair (bp) cDNA sequence of phosphopyruvate hydratase was obtained from the muscles of Marsupenaeus japonicus using RT-PCR and RACE. The results suggested that enolase is highly expressed in the muscle of M. japonicus but not in other organs. We compared the nucleotide sequence of enolase in M. japonicus to that in other species and found 93, 49.08, 47.1, and 44.62% homology to the enolase of Penaeus monodon, Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio, respectively. Furthermore, the amino acid sequence of M. japonicus showed 97, 77, 73, and 69% homology to the corresponding sequences in the other four species, respectively. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus infection dramatically altered the expression level of enolase. Knockdown of enolase resulted in a significant up-regulation (P<0.01) of immune-related genes such as l-lectin, mitogen-activated protein kinase (MAPK), nitric oxide synthase (NOS), prophenoloxidase (proPO), and signal transducer and activator of transcription (STAT), and a significant down-regulation of p53 and TNF (P<0.01) in the muscle of shrimp. We first used RNAi to demonstrate that enolase is very important for WSSV infection of kuruma shrimp, and reported that the time of death was delayed in WSSV-infected shrimp. Characterization of M. japonicus enolase will enable us to further study the role of this enzyme on the immune function in shrimp. Statement of relevancePhosphopyruvate hydratase (enolase) is a housekeeping gene for energy production in many eukaryotes. The 1914bp cDNA sequence of phosphopyruvate hydratase was obtained from the muscles of shrimp Marsupenaeus japonicus. The results suggested phosphopyruvate hydratase was highly expressed in the muscle transcriptome of M. japonicus but not in other organs. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus infection will changed phosphopyruvate hydratase expression tempestuously after the challenge. When phosphopyruvate hydratase was knock down, immune genes like L-lectin, MAPK, NOS, proPO and STAT were up-regulated significantly (P<0.01), but p53 and TNF were down-regulated significantly (P<0.01) in the muscle of shrimp. We firstly demonstrated that phosphopyruvate hydratase is very important for the progress of WSSV infection and the dead time of WSSV-infected shrimp was significantly delayed by the RNAi of phosphopyruvate hydratase. Characterization of M. japonicus phosphopyruvate hydratase will enable us to further study on the role of phosphopyruvate hydratase on immune function in shrimp. Our study is very related to the antiviral immunity of shrimp and belong to the disease of aquaculture.